Growth of endothelial cells on microfabricated silicon nitride membranes for anin vitro model of the blood-brain barrier

Abstract

The blood-brain barrier (BBB) is composed of the brain capillaries, which are lined by endothelial cells displaying extremely tight intercellular junctions. Several attempts at creating anin vitro model of the BBB have been met with moderate success as brain capillary endothelial cells lose their barrier properties when isolated in cell culture. This may be due to a lack of recreation of thein vivo endothelial cellular environment in these models, including nearly constant contact with astrocyte foot processes. This work is motivated by the hypothesis that growing endothelial cells on one side of an ultra-thin, highly porous membrane and differentiating astrocyte or astrogliomal cells on the opposite side will lead to a higher degree of interaction between the two cell types and therefore to an improved model. Here we describe our initial efforts towards testing this hypothesis including a procedure for membrane fabrication and methods for culturing endothelial cells on these membranes. We have fabricated a 1 μm thick, 2.0 μm pore size, and ∼55% porous membrane with a very narrow pore size distribution from low-stress silicon nitride (SiN) utilizing techniques from the microelectronics industry. We have developed a base, acid, autoclave routine that prepares the membranes for cell culture both by cleaning residual fabrication chemicals from the surface and by increasing the hydrophilicity of the membranes (confirmed by contact angle measurements). Gelatin, fibronectin, and a 50/50 mixture of the two proteins were evaluated as potential basement membrane protein treatments prior to membrane cell seeding. All three treatments support adequate attachment and growth on the membranes compared to the control.